**4.2 Enzymatic treatments**

Proteases hydrolyse the peptide linkages between the amino acid units of proteins. Protease activity exists in grape berries [100, 101] and yeast [101–108] as described by several authors. One important aspect is their potential role in wine protein haze reduction [90, 109]; however, proteases have low activity concerning haze-forming proteins, which consequently persist during the vinification process. It is essential, that proteases have to be active under specific wine conditions, namely acid pH, the existence of ethanol, sulphites, phenolics and if possible act at low temperatures. One more challenge is the resistance of PR proteins against proteolysis due to their molecular features such as disulphide bonds and glycosylations. However, proteases from plants (papain from papaya, bromelain from pineapple) have been tested with some promising results concerning their effectiveness in the degradation of heat-unstable proteins from white wine [110–113]. However, the search for fungal enzymes that could degrade wine proteins has so far remained ineffective [114]. As unfolded proteins are more easily cleaved by enzymes, the subsequent phase was the evaluation of the mutual effects of protease addition and heat treatment. Heat treatment joint with the application of proteolytic enzyme can decrease the formation of white wine protein instability; however, the low specificity of commercially disposable proteases for the haze-forming proteins seems to decrease significantly the possibilities of offering this strategy as shown by Pocock et al. [91]. A fungal acid protease resulting from *Aspergillus* sp. rich in aspergillopepsin I (EC 3.4.23.18) and aspergillopepsin II (or aspergilloglutamic peptidase, EC 3.4.23.19) in association with flash pasteurisation (75°C) of the grape juice was confirmed to eliminate haze-forming proteins and consequently stabilises the wines [92, 93]. The application of aspergillopepsin I to eliminate haze-forming proteins in grape must and wine is already authorised by the International Organisation of Vine and Wine [115] Resolution OIV-OENO 541A-2021 and Resolution OIV-OENO 541B-2021. Aspergillopepsin is active at juice and wine pH and at a temperature greater than the melt temperature of haze-forming proteins (chitinases and TLPs, 56 and 62°C, respectively). Therefore, after application of aspergillopepsin I, one short-term heating (60 and 75°C; 1 minute) must be performed as it contributes to the unfolding of haze-forming proteins and facilitates their enzymatic degradation by proteases, as well as leads to denaturation of the protease itself [115], Resolution OIV-OENO 541A-2021; Resolution OIV-OENO 541B-2021. In this context, a protease of *Botrytis cinerea* BcAp8 has been described to hydrolyse grape chitinases at moderate temperatures [116]. Also, evaluation of the effects of the joint use of heat treatment (75°C, 2 minutes) and application of proteases on the protein stability was recently studied by Comuzzo et al. [117]. These authors also evaluated the effect of the heat treatment with application of protease on the wine volatile composition and observed that the wines submitted to this treatment presented a lower content of esters produced during alcoholic fermentation and a higher concentration of esters that are characteristic of ageing such as ethyl lactate [117]. The potential of ultrafiltration (UF), in association with heat and proteolytic enzymes,

to eliminate haze-forming proteins and stabilise white wine was evaluated by Sui et al. [90]. Since the treatment with enzymes (proteases) to eliminate wine hazeforming proteins needs a previous thermal treatment to denaturant them, recently the application of ultra-high-pressure homogenisation (UHPH) was suggested as a possible alternative to the heat treatment. In this way, the application of UHPH could be in the future a new technological solution for using enzymes in the wine protein stabilisation process and probably with a lower impact on the wine volatile composition [118].

### **4.3 Fining and adsorption treatments**

These practices include the use of adsorbents [117], such as zirconium dioxide (ZrO2) also known as zirconia [37, 119–121] carrageenan [6, 92, 122], silica gel, hydroxyapatite and alumina [42], magnetic nanoparticles [123] zeolites [124, 125] and dicarboxymethyl cellulose [126]. However, all of them are at the moment under investigation and therefore not allowed by the International Organisation of Vine and Wine (OIV) or by the European Union (EU) legislation for application in wine.

Mannoproteins [127] are already allowed to be used by the OIV [115]. Chitin and chitosan [127, 128] have been authorised by the European Union (EU) for removal of contaminant and heavy metals, avoidance of turbidity and decrease of unwanted *Brettanomyces* spp. population (EU) 53/2011), but only chitin (Oeno 367-2009 Chitin-Glucan [115] and chitosan (Oeno 368-2009 Chitosan [115] from the cell walls of *Aspergillus niger* or *Agaricus bisporus* are allowed to be applied in wine.

In recent times, some researchers also studied the application of nanomaterials to remove unstable wine proteins [129]. Magnetic steel nanoparticles coated with acrylic acid have been experimented for the selective removal of pathogenesisrelated proteins from wines by cation exchange mechanism due to the existence of carboxylic acid groups in the modified surface, and the results showed that they are highly efficient in decreasing haze-forming proteins [122, 130, 131]. Although these nanoparticles have been found to be effective in removing proteins in proteinunstable wines, their efficiency in wines seems to be affected by the low pH of wines that affects the cation exchange capacity of the nanoparticles due to the protonation of the carboxylic acid groups. Also, mesoporous nanomaterials proved to have high efficiency in decreasing haze-forming proteins with lesser wine aroma decrease compared with bentonite fining [132].

Wine-unstable proteins could also be adsorbed by zirconium dioxide [4, 119, 120, 133], a metal oxide usually known as zirconia, and consequently stabilise the wine by removing, especially, wine protein fractions between 20 and 30 kDa. Also, zirconium oxide pellets enclosed into metallic cage submerged in wine at 25 g/L for 72 hours stabilised white wines by removing unstable proteins with the advantage to be regenerated [37].

Results show that the water-insoluble dicarboxymethyl cellulose successfully reduced the wine protein content and turbidity, producing heat-stable wines with concentrations higher than 0.25 g/L [126].

Polysaccharides extracted from seaweeds were also studied by several researchers due to their negative charge at low pH, can electrostatically flocculate and precipitate positively charged proteins and remove wine unstable proteins [6, 122, 134]. Carrageenan uses at different winemaking stages were considered, and the application stage showed to be very important for its effectiveness [6, 92] More recently, Arenas et al. [17] showed that k-carrageenan reduced the content of pathogen-related proteins and consequently the wines protein instability, being even more efficient than sodium and calcium bentonites (**Figure 1**). On the other hand, these authors also showed that chitosan from fungal origin was unable to

*White Wine Protein Instability: Origin, Preventive and Removal Strategies DOI: http://dx.doi.org/10.5772/intechopen.101713*

#### **Figure 1.**

*Reversed-phase HPLC results and percentage reduction of turbidity (NTU), total protein (mg/L), Vitis vinifera thaumatins (VVL, mg/L) and chitinase (mg/L) for Albariño white wine produced without prefermentative skin maceration and the impact of the different products applied for its protein stabilisation. Control wine without any additive; after addition of k-carrageenan (100 g/hL); after addition of sodium bentonite (120 g/hL); after addition of calcium bentonite (120 g/hL). All chromatograms were acquired by analysis of a 5 mg/mL solution of the high molecular weight after elimination of the low-molecular-weight material by application of 6 M urea and repeated ultrafiltration through a 10 kDa cut-off membrane (adapted from Arenas et al. [17]).*

heat stabilise the wines, and it was also observed that after the application of this oenological product, the levels of pathogen-related proteins remained unchanged. Additionally, the application of the fungal chitosan decreased the concentration of wine polysaccharides by 60%, as also observed after the application of sodium and calcium bentonite (16–59%). However, the application of k-carrageenan did not change the concentration of wine polysaccharides.

Chitin [135] and chitosan [128], polysaccharides mainly from *Aspergillus niger*, also have the capacity to decrease wine haze-forming proteins. It was observed that wine haze induced by the heat test is reduced by 50% after the addition of 1 g/L of chitin, while the addition of 20 g/L of chitin decreased the haze by 80%. The haze decrease perceived was related to the removal of the class IV grape chitinases [136]. Colangelo et al. [128] also showed that wines fined with 1 g/L of fungal chitosanglucan enhanced heat stability at 55−62°C, and this was also due to the reduction of chitinases.

Mannoproteins existing in yeast cell walls have also been reported to have a protective effect on wine protein haze development [137, 138]. Waters et al. [54] showed that mannoproteins protect unstable wine proteins, avoiding wine turbidity when wine is exposed to high temperatures; these authors indicated that this action does not avoid the protein precipitation. Instead, they detected a reduction in particle size, justifying, in this way, the wine stabilisation observed when determined

by turbidimetry. However, their effectiveness for protein stabilisation is highly dependent on the mannoprotein structural characteristic, according to Ribeiro et al. [137], the effectiveness of commercial mannoproteins was related to their chemical composition, namely their high mannose-to-glucose ratio.
